As a consequence of a worldwide vaccination effort, smallpox as a naturally occurring disease was eradicated in the late 1970s. The threat that variola virus (VARV), the causative agent of smallpox, may be accidentally or maliciously released has led to new interest in vaccinating the military and other “first responders” against orthopoxviruses. This renewed interested in vaccination is further supported by the potential that bioinformatics coupled with synthetic biology could be used to engineer orthopoxvirus-based bioterrorism weapons. This latter threat is substantiated by recent and ongoing studies identifying the subtle genetic differences between orthopoxviruses, in particular VARV, which impact pathogenesis and viral tropism [ref. 1-3]. Additionally, naturally occurring monkeypox is an emerging zoonosis [ref. 4, 5]. Despite being localized to regions of Africa, a monkeypox outbreak recently occurred in the United States [ref. 6], representing the potential for worldwide dissemination of this orthopoxvirus.
The current licensed orthopoxvirus vaccine, Dryvax, and the newly licensed ACAM2000 consist of live vaccinia virus (VACV) [ref. 7-9]. These vaccines are highly protective and give long-lasting immunity to the vaccinated individual. However, the use of live VACV is associated with a multitude of health risks. These risks range from the potential of spreading the virus to other sites on the body, including the eye, and to non-vaccinated persons in close contact with the vaccinee [ref. 7-9]. More serious and life-threatening risks include encephalitis, progressive vaccinia, eczema vaccinatum, myocarditis, and even death [ref. 8]. Because of these health risks, vaccination is contraindicated in pregnant women, the immuno-compromised, and in persons with dermatological abnormalities, such as eczema [ref. 7-9]. Kretzschmar, M., et al recently reported that the frequency of death associated with vaccination might be higher than previously believed [ref. 10]. To diminish these heath risks, attenuated VACV viruses, such as modified vaccinia Ankara and LC8 m16 have been developed [ref. 11-14]. However, these attenuated viruses fail to induce protective immunity in immuno-compromised animals, possibly due to host defects in B-cell antibody class switching [ref. 15]. Furthermore, attenuated viruses still encode a multitude of proteins, many of which are involved in immune modulation or have unknown functions. The potential risk of these factors remains largely unexamined.
As an alternative to live virus vaccines, DNA and/or protein-based-subunit vaccines targeting one or more orthopoxvirus immunogens are being explored. Early studies demonstrated that protein or DNA-expressing A33 or B5 could protect mice from VACV challenge [ref. 16]. Recently, it was reported that vaccination with the A33 protein protects mice from challenge with ectromelia virus [ref. 17]. Currently, targets of orthopoxvirus subunits vaccines include D8, H3, A33, A27, L1 and B5 [ref. 16-24]. We developed a combination DNA vaccine (termed 4pox) that targets four orthopoxvirus antigens (L1, A27, B5 and A33) [ref. 21-23]. Orthopoxvirus has two antigenically distinct infectious forms, extracellular enveloped virions (EEV) and intracellular mature virions (IMV) [ref. 25]. EEV particles are primary involved in viral dissemination within an infected host, while the more environmentally stable IMV are thought to be involved in spread between hosts. Accordingly, our 4pox vaccine targets multiple proteins on both infectious forms of orthopoxviruses, the IMV (L1 and A27) and the EEV (B5 and A33). Plasmids expressing these genes elicit antibody responses against each protein when delivered to the skin by gene-gun or electroporation [ref. 20-23]. Importantly, the 4pox vaccine can protect mice and non-human primates from lethal challenge with VACV or monkeypox virus, respectively [ref 21-23]. Fogg, C., et al. demonstrated that a protein vaccine consisting of these targets can also protect animals from lethal orthopoxvirus challenges [ref. 24]. Thus, these combinations of orthopoxvirus targets are effective and valuable targets for a subunit orthopoxvirus vaccine.
The L1 protein is encoded by the L1R gene and is a target of the 4pox vaccine [ref. 21-23]. L1 is a myristylated 23-29 kDa membrane protein located on the surface of IMVs and beneath the envelope on EEVs [ref. 26, 27]. This molecule is highly conserved among the orthopoxviruses. Importantly, the L1 protein is a target of potently neutralizing antibodies [ref. 20-23, 28-31], making it an attractive target for vaccines and therapeutics. By “neutralizing antibody” it is meant an antibody (or antibody fragment such as an F(Ab′)2 fragment) that has a mechanism of action of specifically interacting with and binding to a viral target molecule (e.g. L1) and this interaction prevents the virus from being able to productively infect a target cell by means such as (but not limited to), preventing receptor binding, preventing important conformational changes in a virus molecule (directly or indirectly) required to infect a target cell or by preventing an important cellular signaling event needed for infection.
The precise function(s) of L1 remains to be characterized because deletion is a lethal [ref. 27]. However, in the absence of L1, particle morphogenesis and formation of infectious virus is blocked, suggesting a role for L1 in IMV assembly [ref. 27]. Antibodies against L1 can neutralize viral infectivity, suggesting that L1 may also play a role in particle entry either directly or indirectly [ref. 30]. The structure of L1 has been solved and reveals a molecule comprised of a bundle of α-helices packed against a pair of two-stranded β-sheets, held together by four loops [ref. 32]. The structure also contains three disulfide bonds that are formed in the cytoplasm by a virus-encoded disulfide bond formation pathway [ref. 33]. These disulfide bonds are critical for the interaction of potently neutralizing antibodies [ref. 30]. Indeed, the crystal structure of L1 bound by a potently neutralizing antibody MAb-7D11 was recently reported [ref. 34]. This structure revealed that potentially neutralizing antibodies bind to a discontinuous epitope consisting of two loop regions held together by a disulfide bond.
It remains a problem that the current live-orthopoxvirus vaccine is associated with minor to serious adverse affects, and is contraindicated for use in a significant portion of the population. As an alternative vaccine, we have previously shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. [Refs. 21 and 22] Because antibodies are critical for protection against secondary orthopoxvirus infections [ref. 35-37], we were interested in strategies that will enhance the humoral immune response against vaccine targets. To that end, we were interested in enhancing the 4pox DNA vaccine such that it will require only one or two vaccinations to elicit protection in the vaccinated host.
Another problem was encountered when we attempted to reduce the number of cartridges by co-delivery of two antigens (L1 and A33) conjugated on the same carrier particle for gene gun DNA vaccination. For multi-pox DNA vaccination, it is desirable to reduce the number of cartridges required for each vaccination, so that the capacity to deliver two or more antigens in one cartridge would be ideal. However, we discovered that when we mixed plasmids expressing L1R and A33R (e.g., precipitated on the same gold particle for gene gun delivery), there was a good response against A33 protein but essentially no response to the L1 protein (no neutralizing antibody). This is believed to be due to interference of the L1 and A33 with each other. [ref. 20]